1. Field of the Invention
The present invention relates generally to the field of in vitro diagnostics. More particularly, the present invention relates to the measurement of attomole concentrations in biological assays within nanoliter-to-subnanoliter scale fluid volumes using such diagnostics.
2. Description of the Prior Art
The current in vitro diagnostics (IVD) market has been estimated to be eighteen billion dollars (U.S.) annually. The key market segments are immunoassay and clinical chemistry, with the immunoassay market being much more profitable for IVD companies.
In today's market, automated immunoassays can detect concentrations in the low attomole range; can provide results in from twenty to thirty minutes; and can be produced at low cost, namely, $0.35 per test. Current assays can give results using several hundred microliters of specimen and achieve low attomole detection using chemiluminescence or fluorescence detection in a solid-phase format. In order to develop an ultrasmall device for the same assay using only tens to hundreds of nanoliters of sample, depending upon analyses concentration, sensitivity of detection will need to be significantly improved. This is particularly the case for immunoassays as no equivalent of PCR (polymerase chain reaction) exists for the target amplification of an antigen. Therefore, very sensitive and inexpensive detection systems must be developed for point-of-care devices.
A central challenge, then, is to increase the sensitivity and accuracy of immunoassays within nanoscale volumes. Since the higher sensitivity systems employ high surface area solid-phase systems with light signals for detection, an alternative approach may be made with fluorescence immunoassays using particles. Many fluorophore labels, lasers and light collection systems have been developed to increase fluorescent assay sensitivity. Among the important strategies used to enhance sensitivity and accuracy are the reduction of background fluorescence using longer-lived fluorophores combined with time resolution of fluorescence detection. In addition, fluorophores emitting at a longer wavelength may be used so that light of shorter wavelength from common background fluorescence sources may be filtered out. Finally, materials with minimal fluorescence may be used in the analysis chambers and filters.
These efforts presuppose that care is taken to eliminate background binding from non-specific adsorption or entrainment from incomplete washing of the fluorescent labeled antibody or antigen. Regardless of the care taken to eliminate stray light or the inherent fluorescence of material on the surface or in solution, if the signal being measured is present due to an errant binding of the labeled antigen or antibody, these efforts will be for naught. Moreover, economic and technical drawbacks and limitations also exist for the alternative strategies noted in the preceding paragraph.
Clearly, a technique by which the signal-to-noise ratio for measurement of the foregoing type in nanoscale volumes could be improved would have great benefit in the IVD market. Such a technique is provided by the present invention.